Nuclear Magnetic Resonance Studies on Nitrophorins and Paramagnetic Model Complexes

Persistent Link:
http://hdl.handle.net/10150/195238
Title:
Nuclear Magnetic Resonance Studies on Nitrophorins and Paramagnetic Model Complexes
Author:
YANG, FEI
Issue Date:
2010
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The majority of the work in this dissertation describes the characterization of the nitrophorins (NPs) by NMR spectroscopy.NP7 was studied and compared to NP2 and relevant mutants.NP7 is shown to have three additional amino acids at the N-terminus, which contribute significantly to the protein fold stability and maintenance of NP7 function. The paramagnetic form of the protein has large 1H NMR chemical shifts and broad signals due to protein aggregation. The 1H NMR and CD spectroscopy both revealed that NP7 has mainly the A heme orientation, and the mutation of Glu27 to Val27 leads to an A:B ratio change, from mainly B to mainly or almost exclusively A.NMR investigation of the ferriheme resonances of the low-spin complexes of NP2 and mutant NP2(V24E) with imidazole (ImH), histamine (Hm) and cyanide (CN-) ligands as a function of pH has been completed. Strong chemical exchange cross peaks were observed in the NOESY/EXSY spectra at low pH (pH* 5.5 to 4.0) for the three wt NP2 complexes, which indicate an interchange between two ruffling distortions of the heme. A dramatic change of A:B ratios with pH was observed for the three NP2(V24E) ligand complexes. They both are believed to be a result of a change in protein structure near E53 when it is protonated at low pH.Homo-dimers of NP1 and NP4 were investigated. 1H{15N}-HSQC NMR experimental results revealed that holo-NP4 is a homo-dimer at low pH and a mixture of dimer and monomer at high pH. In contrast, holo-NP4-Hm is monomeric at high pH (6.5 and 7.5). A H-bond between Asp30 and the Hm ligand is responsible for destabilization of the homo-dimer. NP1 was also shown to be a mixture of dimer and monomer at high pH.Heme d1 model complexes were studied. The high-spin form and low-spin forms of the Fe(III) complexes with 2-methylimidazole or imidazole, for monooxo-octaethylchlorin, trans-dioxo-octaethylisobacteriochlorin and 2,7-dioxo-octaethyliso- bacteriochlorin, were characterized by NMR spectroscopy. Spin density maps that show the orbital used for spin delocalization have been generated from Hückel calculations.Preliminary results of additional studies of the nitrophorins and future directions of study are presented.
Type:
text; Electronic Dissertation
Keywords:
heme; nitrophorin; NMR; paramagnetic
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Walker, F. Ann
Committee Chair:
Walker, F. Ann

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleNuclear Magnetic Resonance Studies on Nitrophorins and Paramagnetic Model Complexesen_US
dc.creatorYANG, FEIen_US
dc.contributor.authorYANG, FEIen_US
dc.date.issued2010en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe majority of the work in this dissertation describes the characterization of the nitrophorins (NPs) by NMR spectroscopy.NP7 was studied and compared to NP2 and relevant mutants.NP7 is shown to have three additional amino acids at the N-terminus, which contribute significantly to the protein fold stability and maintenance of NP7 function. The paramagnetic form of the protein has large 1H NMR chemical shifts and broad signals due to protein aggregation. The 1H NMR and CD spectroscopy both revealed that NP7 has mainly the A heme orientation, and the mutation of Glu27 to Val27 leads to an A:B ratio change, from mainly B to mainly or almost exclusively A.NMR investigation of the ferriheme resonances of the low-spin complexes of NP2 and mutant NP2(V24E) with imidazole (ImH), histamine (Hm) and cyanide (CN-) ligands as a function of pH has been completed. Strong chemical exchange cross peaks were observed in the NOESY/EXSY spectra at low pH (pH* 5.5 to 4.0) for the three wt NP2 complexes, which indicate an interchange between two ruffling distortions of the heme. A dramatic change of A:B ratios with pH was observed for the three NP2(V24E) ligand complexes. They both are believed to be a result of a change in protein structure near E53 when it is protonated at low pH.Homo-dimers of NP1 and NP4 were investigated. 1H{15N}-HSQC NMR experimental results revealed that holo-NP4 is a homo-dimer at low pH and a mixture of dimer and monomer at high pH. In contrast, holo-NP4-Hm is monomeric at high pH (6.5 and 7.5). A H-bond between Asp30 and the Hm ligand is responsible for destabilization of the homo-dimer. NP1 was also shown to be a mixture of dimer and monomer at high pH.Heme d1 model complexes were studied. The high-spin form and low-spin forms of the Fe(III) complexes with 2-methylimidazole or imidazole, for monooxo-octaethylchlorin, trans-dioxo-octaethylisobacteriochlorin and 2,7-dioxo-octaethyliso- bacteriochlorin, were characterized by NMR spectroscopy. Spin density maps that show the orbital used for spin delocalization have been generated from Hückel calculations.Preliminary results of additional studies of the nitrophorins and future directions of study are presented.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjecthemeen_US
dc.subjectnitrophorinen_US
dc.subjectNMRen_US
dc.subjectparamagneticen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWalker, F. Annen_US
dc.contributor.chairWalker, F. Annen_US
dc.contributor.committeememberWalker, F. Annen_US
dc.contributor.committeememberLichtenberger, Dennisen_US
dc.contributor.committeememberZheng, Zhipingen_US
dc.contributor.committeememberGhosh, Indraneelen_US
dc.contributor.committeememberJacobsen, Neilen_US
dc.identifier.proquest10825en_US
dc.identifier.oclc752260944en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.